Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A(2) class

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Crystal structure of a myotoxic Asp49-phospholipase A(2) with low catalytic activity: Insights into Ca2+ -independent catalytic mechanism

A myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu) and other Asp49-PLA(2)s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA(2)s, making a hydrogen bond with the atom 0 delta 2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA(2)s which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA(2)s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA(2)s. (c) 2008 Elsevier B.V All rights reserved.

Crystal structure of a phospholipase A(2) homolog complexed with p-bromophenacyl bromide reveals important structural changes associated with the inhibition of myotoxic activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

At the interface: Crystal structures of phospholipases A(2)

The protein content of many snake venoms often includes one or more phospholipases A(2) (PLA(2)). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA(2)-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA(2)s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA(2)s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA(2)s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49-PLA(2)s and propose a model to explain the Ca2+ independent membrane damaging activity. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Crystal structure of a dimeric Ser49 PLA(2)-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis venom

Zhaoermiatoxin, an Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA(2)-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49 -> Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA(2) homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA(2) catalytic activity. A single crystal with maximum dimensions of 0.2 x 0.2 x 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 angstrom using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6(4), with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 angstrom.

A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A(2)S

Venom phospholipase A(2)s (PLA(2)s) display a wide spectrum of pharmacological activities and, based on the wealth of biochemical and structural data currently available for PLA(2)S, mechanistic models can now be inferred to account for some of these activities. A structural model is presented for the role played by the distribution of surface electrostatic potential in the ability of myotoxic D49/K49 PLA(2)s to disrupt multilamellar vesicles containing negatively charged natural and non-hydrolyzable phospholipids. Structural evidence is provided for the ability of K49 PLA(2)s to bind phospholipid analogues and for the existence of catalytic activity in K49 PLA(2)s. The importance of the existence of catalytic activity of D49 and K49 PLA(2)s in myotoxicity is presented. (C) 2003 Elsevier Ltd. All rights reserved.

Crytallization and high-resolution X-ray diffraction data collection of an Asp49 PLA(2) from Bothrops jararacussu venom both in the presence and absence of Ca2+ ions

Snake venom PLA(2)s have been extensively studied due to their role in mediating and disrupting physiological processes such as coagulation, platelet aggregation and myotoxicity. The Ca2+ ion bound to the putative calcium-binding loop is essential for hydrolytic activity. We report the crystallization in the presence and absence of Ca2+ and X-ray diffraction data collection at 1.60 Angstrom (with Ca2+) and 1.36 Angstrom (without Ca2+) of an Asp49 PLA(2) from Bothrops jararacussu venom. The crystals belong to orthorhombic space group C222(1). Initial refinement and electron density analysis indicate significant conformational. changes upon Ca2+ binding. (C) 2004 Elsevier B.V. All fights reserved.

Crystal structure of a novel myotoxic Arg49 phospholipase A(2) homolog (zhaoermiatoxin) from Zhaoermia mangshanensis snake venom: Insights into Arg49 coordination and the role of Lys122 in the polarization of the C-terminus

The venom of Zhaoermia mangshanensis, encountered solely in Mt Mang in China's Hunan Province, exhibits coagulant, phosphodiesterase, L-amino acid oxidase, kallikrein, phospholipase A(2) and myotoxic activities. The catalytically inactive PLA(2) homolog referred to as zhaoermiatoxin is highly myotoxic and displays high myonecrotic and edema activities. Zhaoermiatoxin possesses a molecular weight of 13,972 Da, consists of 121 amino-acid residues crosslinked by seven disulfide bridges and shares high sequence homology with Lys49-PLA(2)s from the distantly related Asian pitvipers. However, zhaoermiatoxin possesses an arginine residue at position 49 instead of a lysine, thereby suggesting a secondary Lys49 -> Arg substitution which results in a catalytically inactive protein. We have determined the first crystal structure of zhaoermiatoxin, an Arg49-PLA(2), from Zhaoermia mangshanensis venom at 2.05 A resolution, which represents a novel member of phospholipase A(2) family. In this structure, unlike the Lys49 PLA(2)s, the C-terminus is well ordered and an unexpected non-polarized state of the putative calcium-binding loop due to the flip of Lys122 towards the bulk solvent is observed. The orientation of the Arg-49 side chain results in a similar binding mode to that observed in the Lys49 PLA(2)s; however, the guadinidium group is tri-coordinated by carbonyl oxygen atoms of the putative calcium-binding loop, whereas the N zeta atom of lysine is tetra-coordinated as a result of the different conformation adopted by the putative calcium-binding loop. (c) 2008 Elsevier Ltd. All rights reserved.

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA(2)) and II (Asp49 PLA(2)) and their possible blockage by indomethacin. BthTX-1 (5 mu g/ml) and BthTX-II (5 mu g/ml) increased perfusion pressure (PP; ct(120) = 110.28+/-3.70 mmHg; BthTX I = 171.28+/-6.30* mmHg; BthTX II = 175.50+/-7.20* mmHg), renal vascular resistance (RVR; ct(120) = 5.49+/-0.54 mmHg/ml.g(-1) min(-1); BthTX I = 8.62+/-0.37* mmHg/ml g(-1) min(-1); BthTX II=8.9+/-0.36* mmHg/ml g(-1) min(-1)), urinary flow (UF; ct(120)= 0.14+/-0.01 ml g(-1) min(-1); BthTX I=0.32+/-0.05* ml g(-1) min(-1); BthTX II=0.37+/-0.01* ml g(-1) min(-1)) and glomerular filtration rate (GFR; ct(120)=0.72+/-0.10 ml g(-1) min(-1); BthTX I=0.85+/-0.13* ml g(-1) min(-1); BthTX II=1.22+/-0.28* ml g(-1) min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa+; ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK+;ct(120)=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA(2). In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA(2). (C) 2005 Elsevier Ltd. All rights reserved.

Crystal structure of myotoxin-II: A myotoxic phospholipase A2 - Homologue from Bothrops moojeni venom.

The crystal structure of Myotoxin-II (MjTX-II), a Lys49 PLA(2)-homologue from Bothrops moojeni venom has been determined and refined at 2.0 Angstrom to a crystallographic residual of 19.7% (R-free = 28.1%). MjTX-II is a dimer in the crystal, with the monomers in the asymmetric unit related by a two-fold symmetry axis running through the dimer interface. The dimers of MjTX-II and the Lys49 PLA(2) from B. asper venom are similar, however the relative orientations of the monomers suggests a flexible dimer interface, which serves as a hinge between the two molecules.